MA3-006

antibody from Invitrogen Antibodies

Targeting: HSPA4 HS24/P52, HSPH2

Provider product page for MA3-006

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [75]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [5]
Immunohistochemistry [3]
Other assay [20]

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Product number: MA3-006 - Provider product page
Provider: Invitrogen Antibodies
Product name: HSP70 Monoclonal Antibody (3A3)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: MA3-006 detects several members of the heat shock protein 70 kDa (HSP70) gene family including HSP70, HSC70, p75, and following heat shock, HSP72 from yeast, Drosophila, fish, porcine, plant, mouse, avian, arthropod (red claw crayfish & black tiger prawn), amphibian, non-human primate, and human samples.
Antibody clone number: 3A3
Concentration: 1 mg/mL

HSPA4 protein structure - MA3-006 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Hsp70 was performed by loading 20 µL of gill tissue lysates from the salt marsh mussel, Guekensia demissa, isolated from various coves in Rhode Island (indicated above the lanes) and processed by homogenization in 32mM Tris-HCl, 1mM EDTA, and 2% SDS homogenization buffer, per well onto an SDS-PAGE gel. Proteins extracted from the supernatants were transferred to nitrocellulose membrane and blocked overnight with 5% milk in TBST. The membrane was probed with an Hsp70 monoclonal antibody (Product # MA3-006) at a dilution of 1:1000, washed in TBST, and probed with an HRP-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1:3000. Detection was performed using SuperSignal West Pico (Product # 34080). Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-HSP70 Monoclonal Antibody (3A3) (Product # MA3-006) and a 70kDa band corresponding to HSP70 was observed across cell lines and tissues tested and was induced upon heat shock in Jurkat cell line. Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), A549 (Lane 2), IMR-32 (Lane 3), Jurkat (Lane 4), Jurkat treated with heat shock (42deg for 30 min followed by 37deg for 3 hours) (Lane 5), MDCK (Lane 6), NIH/3T3 (Lane 7), PC-12 (Lane 8), COS-7 (Lane 9) and Mouse Ovary (Lane 10) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Heat Shock Protein 70 (Hsp70) was performed by loading 50 µg of the indicated whole cell lysates and 15 µL of PageRuler Prestained Protein Ladder (Product # 26616) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Hsp70 monoclonal antibody (Product # MA3-006) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween 20, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:20,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) (Product # MA3-006) shows staining in NIH-3T3 cells. Heat Shock Protein 70 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Heat Shock Protein 70 (Product # MA3-006) at a dilution of 1:100-1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) (Product # MA3-006) shows staining in HeLa cells. Heat Shock Protein 70 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Heat Shock Protein 70 (Product # MA3-006) at a dilution of 1:100-1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) (Product # MA3-006) shows staining in MCF-7 cells. Heat Shock Protein 70 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Heat Shock Protein 70 (Product # MA3-006) at a dilution of 1:100-1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Heat Shock Protein 70 (Hsp70) (green) in HeLa and NIH3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a Hsp70 monoclonal antibody (Product # MA3-006), at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat-anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of HSP70 was performed using 70% confluent log phase HEK-293 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with HSP70 Monoclonal Antibody (3A3) (Product # MA3-006) at 1:200 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing localization to nucleus and cytoplasm. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized human Testis tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (Product # MA3-006) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized human Tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (Product # MA3-006) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized human Breast carcinoma tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:50 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (Product # MA3-006) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 2 Hsp70 was induced in P . viridis by heat shock. Protein samples from the adductor muscle (A, B), foot (C, D), gill (E, F) and mantel (G, H) were resolved in 7% SDS polyacrylamide gels and either stained with Coomassie blue (A, C, E, G) or blotted to membranes and probed with antibody to Hsp70 (B, D, F, H). Arrows labeled p70 indicate the position of 70 kDa proteins in the gel. M, molecular mass markers in kDa; H-Hsp70, recombinant human Hsp70.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 1 Gadkin is expressed in lymphoid organs and in immune cells. A. Lysates of WT and Gadkin -/- (labeled as KO) tissues were analyzed by immunoblotting using Gadkin- and AP-1-specific antibodies. B. Lysates of WT and Gadkin -/- thymus, of DCs derived from WT and Gadkin -/- bone marrow, as well as of B lymphocytes (i.e. B220-positive B cells) and T lymphocytes (i.e. CD3-positive T cells) isolated from spleen were analyzed by immunoblotting using Gadkin-specific antibodies. Detection of the constitutively expressed molecular chaperone heat shock cognate protein 70 (Hsc70) was used as loading control. B, B-lymphocytes; T, T-lymphocytes.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 NLHS increased Hsp70 in L. vannamei post-larvae. Protein extracts were resolved by electrophoresis in SDS polyacrylamide gels and either stained with Coomassie Biosafe (A) or blotted to polyvinylidene fluoride membranes and incubated with antibody to Hsp70 (B). Approximately 50 ug of protein was loaded in each lane. 28, non-heat shocked post-larvae heat shock at 30degC, 32degC, 34degC, 36degC and 38degC was for 30 min with recovery at 28degC for 8 h H-Hsp70: Human Hsp70 recombinant protein M: molecular mass standards in kDa. The figure is a representation from two separate experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 NLHS enhanced Hsp70 production in L. vannamei . Post-larvae were exposed to 30 min heat shock from 28degC to 30degC, 32degC, 34degC, 36degC and 38degC, then transferred to 28degC for 8 h. Quantification of Hsp70 was by ELISA. A standard curve, constructed with Human Hsp70 recombinant protein was used to convert ELISA readings obtained with shrimp tissue protein extracts to Hsp70 content. Bars represent the fold difference of Hsp70 quantity in comparison to a non-heated control. The error bars represent the SD from 3 replicates. Asterisks denote statistically significant differences between values obtained for control and heat shocked post-larvae ( P< 0.05). The figure is a representation from two separate experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 4 P . viridis Hsp70 Varied with Recovery Time after Heat Shock. Protein samples from the adductor muscle (A, B), foot (C, D), gill (E, F) and mantel (G, H) were resolved in 7% SDS polyacrylamide gels and either stained with Coomassie blue (A, C, E, G) or blotted to membranes and probed with antibody to Hsp70 (B, D, F, H). Arrows labeled p70 indicate the position of 70 kDa proteins in the gel. M, molecular mass markers in kDa; H-Hsp70, recombinant human Hsp70. C, mussels not receiving heat shock.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 4 Gadkin is regulated in an LPS-dependent manner. A-D. LPS treatment of BMDCs increases Gadkin levels and induces its transient posttranscriptional modification. A. Immature WT BMDCs were either left untreated or incubated for 24 h with LPS. Subsequently, cells were allowed to spread on fibronectin-coated cover slips for 15 min to achieve a more uniform morphology prior to analysis. Cells were processed for immunofluorescence and labeled with Gadkin-specific antibodies. Nuclei were stained with DAPI. Insets show 2x enlarged perinuclear area. Scale bar: 10 mum. B,C. Lysates from WT BMDCs treated with LPS for the indicated times were analyzed by immunoblotting using Gadkin- and Hsc70-specific antibodies. The closely spaced upper band is indicated by red arrows. D. Quantification of immunoblot images like the one depicted in B. Gadkin levels were normalized to Hsc70 levels derived from the same blot. (N = 3 independent experiments, unpaired two-tailed t-test; * = p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of Heat Shock Protein 70 (Hsp70) was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500 µg whole cell lysate with 2 µg of Hsp70 monoclonal antibody (Product # MA3-006) overnight on a rocking platform at 4°C. The immune complexes were captured on 50 µL Protein A/G Plus Agarose (Product # 20421), washed extensively, and eluted with Lane Marker Reducing Sample Buffer (Product # 39000). Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Hsp70 monoclonal antibody (Product # MA3-006) at a dilution of 1:1000 overnight rotating at 4°C, washed in TBST, and probed with goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:20,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Top compounds decrease the expression of Hsp90 clients, and dose-dependently inhibit K-Ras nanoclustering-FRET. ( A - D ) Western blots of HER-2 overexpressing SK-BR-3 ( A ), KRAS mutant triple negative breast MDA-MB-231 ( B ), KRAS mutant pancreatic MIA PaCa-2 ( C ), and KRAS mutant lung HCC-44 ( D ) cancer cell lines. The cells were treated with 0.1% DMSO vehicle control or the indicated concentration of compounds. In total, 2 muM 17-AAG or 10 muM conglobatin A served as the positive control. Note the increase in Hsp90 and, in particular, Hsp70 with the 17-AAG treatment, which was not observed with interface inhibitors. All of the drug treatments were for 24 h; n = 1 independent biological repeat. ( E , F ) K-RasG12V- ( E ) and HRasG12V- ( F ) nanoclustering-FRET in HEK cells co-transfected with mGFP- and mCherry-tagged RasG12V. The cells were treated for 24 h with 0.1% DMSO vehicle control, 2 muM conglobatin A, 2 muM compactin, 0.5 muM FTI277, or the indicated concentrations of x6505 or x1540 ( E ) or 5 muM of these hit compounds ( F ). Compactin and FTI277 block Ras prenylation and farnesylation, respectively, and served as positive controls. The numbers in the bars indicate the number of analyzed cells; n = 3 independent biological repeats. **** p < 0.0001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Quantification of heat shock protein (HSP27) and HSP70 in the hippocampus of APP-overexpressed mice treated with ETAS (200 mg/kg and 1000 mg/kg) and WT mice treated with ETAS (1000 mg/kg) for 1 month, respectively; RFUs: relative fluorescence units; green represents HSP27/70 expression, and blue represents DAPI. Arrows represent fluorescent green staining, indicating positive expression. Magnification is represented x200. HSP27: ** p < 0.01 APP-ETAS (1000 mg/kg) vs. APP-ETAS (200 mg/kg) and APP saline-treated mice; ** p < 0.01 WT-ETAS (1000 mg/kg) vs. WT saline-treated mice; RFUs: relative fluorescence units. Four mice per group and four sections per mouse were used. HSP70: * p < 0.05 APP-ETAS (1000 mg/kg) vs. APP-ETAS (200 mg/kg); ** p < 0.01 APP-ETAS (1000 mg/kg) vs. APP saline-treated mice; ** p < 0.01 WT-ETAS (1000 mg/kg) vs. WT saline-treated mice. RFUs: relative fluorescence units. Four mice per group and four sections per mouse were used.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 6 Two 70 kDa protein bands were resolved in 5% SDS polyacrylamide gels. Tissues corresponding to the adductor muscle, foot, gill and mantel were isolated from P . viridis exposed to NLHS, homogenized, centrifuged and applied to a 5% SDS polyacrylamide gel. Labeled arrows indicate excised regions of the gel subsequently shown by mass spectrometry to contain Pv Hsp70-1 and Pv Hsp70-2. M, molecular mass in kDa; C, mussels not receiving NLHS; HS, mussels receiving NLHS; F, foot; G, gill; MA, mantle; MU, adductor muscle.